Asymmetric capillary for capillary-flow cytometers

ABSTRACT

The present invention provides improved capillaries that lead to increased resolution in conventional capillary-flow cytometers. The cross-sectional shape of capillaries made according to the present invention lack a center of symmetry. In some embodiments, capillaries have inner side walls that are tilted at angles with respect to the collection-system optical axis so that the widest dimension of the inner bore is closest to the collection optical system and have an outer wall closest to the collection optical system with a dimension large enough to minimize the contribution of outer-wall refraction to the collected light signal. Exemplary capillary embodiments include tubes with a rectangular outer wall and a trapezoidal inner wall, a rectangular outer wall and a triangular inner wall, triangular outer and inner walls, a triangular outer wall with a trapezoidal inner wall, and a hemispherical or rhomboid outer wall and trapezoidal or triangular inner wall.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application60/791,002, “Improved Capillary Design for Capillary Flow Cytometers,”which was filed on Apr. 11, 2006, and which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to flow cytometers for countingparticles, and, more particularly, to capillary-flow cytometers.

BACKGROUND OF THE INVENTION

Flow-cytometer systems are used for the detection and counting ofmicro-organisms and for varied applications throughout the life sciencesincluding clinical diagnostics and immunology, protein and nucleic aciddetection, hematology, and oncology. Commercially available instrumentsrange from complex laboratory systems that may be configured for a widerange of measurements to low-cost bench-top systems with more limitedcapabilities. In the current biotechnology market, the price of a flowcytometer typically increases with its measurement precision and withthe number of different measurements it is capable of performing.

Flow cytometers are typically used to identify and count particles withspecific characteristics in a fluid sample. In this disclosure, the term“sample particles” may refer, for example, to latex spheres, bacteria,viruses, DNA fragments, cells, molecules, or constituents of wholeblood. Sample particles may scatter excitation light directly or mayfluoresce when illuminated by light of an appropriate wavelength. Inmany cases, the fluorescent-emission properties are optimized forspecific measurements by attaching probe molecules to the entire sampleparticles or to microscopic structures within the particles.

In a typical flow cytometer, sample particles are transported by aflowing fluid to an excitation volume where they are illuminated withthe focused output beam of a laser or alternative light source. Lightthat is scattered and emitted by the illuminated sample particles iscollected and separated according to emission angle and wavelength usingconventional optical systems. Because the sample particles travelthrough the excitation volume at a high velocity, the light is scatteredand emitted in the form of pulses with amplitudes and temporal profilesthat are determined by the size and shape of the particles, by theirvelocity as they pass through the excitation volume, and by the opticalcharacteristics of the light-collection system. In an ideal case, sampleparticles with the same physical properties produce identical lightpulses. In practice, variations in pulse shape are caused by spatiallydependent variations in sample particle velocity and in collectionefficiency and by the simultaneous illumination of multiple particles.

Light pulses that have been separated according to emission angle andwavelength by the optical system are converted into analog electronicpulses by photomultipliers, solid-state detectors, or alternative lightdetectors. A data-acquisition system is commonly used to convert theanalog signals to a digital data stream for subsequent analysis by adigital signal processor or computer.

The presence of a particular type of sample particle within theexcitation volume is determined by comparing the amplitude of thedetector pulses to fixed reference levels. Errors in the sample-particledetection process are caused by the simultaneous illumination ofmultiple sample particles and by variations in the amplitude and shapeof pulses that are generated by identical sample particles. Theillumination of a single sample particle typically generates asingle-peaked detector pulse that is referred to as a singlet pulse. Thesimultaneous illumination of two sample particles typically generates adetector pulse with two peaks that is referred to as a doublet pulse. Ina typical system, the probability of illuminating more than two sampleparticles is low. Measurement precision and reproducibility aremaximized in systems where individual sample particles pass through theexcitation volume in a sequential fashion and where identical sampleparticles produce pulses with the same shape and amplitude.

FIG. 1 is a schematic representation of a conventional sheath-flowcytometer system 100 in which a sample fluid is surrounded by a sheathfluid that may be in the gaseous or liquid state. The sample is injectedinto the sheath fluid by a core injector 102, and the combined fluidsmove through a flow tube 104 with a smooth, stationary, laminar velocitydistribution that is typically a parabolic function of the radialdistance from the flow-tube axis. Particles in the sample fluid interactwith light from a focused excitation source 106 within an excitationvolume 108 that is downstream from the core injector 102. The diameterof the sample fluid is decreased by gradually reducing the diameter ofthe flow tube 104 in a neckdown region 110 between the core injector 102and the excitation volume 108. In the ideal case, the diameter of thesample fluid in the region of the tube 104 containing the excitationvolume 108 is small enough that cells (or other sample particles) passthrough the excitation volume 108 one at a time. The decreased samplediameter has the added advantages of minimizing radial variations inparticle velocity and in optical collection efficiency.

In the cytometer system 100 of FIG. 1, the sheath fluid is introducedinto a larger-diameter section 112 of the flow tube 104 through apressurized inlet 114. The sample fluid is injected into the surroundingsheath fluid through a pressurized core injector nozzle 102 with an axisthat is typically coincident with the flow-tube axis. The combinedfluids flow through the neckdown region 110 to the excitation volume 108where the sample fluid is illuminated by a focused excitation light beamthat may be generated by a laser, by a laser-driven frequency nonlinearconverter such as a frequency doubler, tripler or quadrupler, by anoptical parametric oscillator, by a light-emitting diode, by asuperluminescent diode, by an arc lamp, or by another light source 106with a suitable combination of brightness and output wavelength.

An excitation optical system 116 is used to concentrate the excitationbeam in the excitation volume 108. The excitation optical system 116 isshown as a simple lens in FIG. 1 but may include one or more componentsselected from the group of conventional diffractive optics, reflectiveoptics, and refractive optics. An optional bandpass filter 118 with hightransmission at the excitation wavelength may be placed between theexcitation light source 106 and the excitation volume 108 to block lightemitted by the excitation source 106 at wavelengths different from theexcitation wavelength.

The focused excitation light interacts with sample particles flowingthrough the excitation volume 108 via several physical processesincluding fluorescence excitation, absorption, small-angle scattering,and large-angle scattering. Sample particles are identified and countedby measuring the wavelength, amplitude, duration, and shape of the lightpulses that are generated when the moving particles are illuminated bythe excitation beam.

Scattered excitation light typically has an angular distribution that isdetermined by the size and shape of the scattering particles. It is,therefore, advantageous to measure the time-dependent amplitude of thelight that is simultaneously scattered at large angles (>45 degrees) andat small angles (<10 degrees) to the excitation-beam propagation axis.Fluorescent light is typically emitted into 4 π solid angle with adistribution that is dependent on the polarization of the excitationlight and, possibly, on other factors.

The signal-to-noise ratio is maximized when the fluorescent andscattered light is viewed against a dark background. In large-anglescatter and fluorescence measurements, the background light level isminimized by collecting light at large angles to the excitation-beampropagation direction and using apertures designed to block non-particlescattered light sources. In forward-scattering measurements, thebackground light level is typically minimized by blocking the excitationbeam.

In the cytometer system 100 of FIG. 1, an optical collection system forlarge-angle light emission 120 gathers fluorescent light and light thatis scattered into a cone of angles around an axis that is orthogonal tothe excitation-beam propagation axis. Scattered light passes through thedichroic beam splitters 122, 124 and is focused onto the active elementof the large-angle scatter detector 126 by a lens 128 or by analternative focusing optical system. Fluorescent light of a firstwavelength is reflected towards a first fluorescence detector 130 by thefirst dichroic beamsplitter 122, and fluorescent light of a second,different, wavelength is reflected by the second dichroic beamsplitter124 towards a second fluorescence detector 132. One or more opticalbandpass filters 134 are typically placed between the excitation volume108 and the detectors 126, 130, 132 to restrict the wavelengths reachingeach detector 126, 130, 132.

Light that is scattered at small angles to the excitation-beampropagation axis is collected by the forward-scatter imaging system 136.A beam block 138 is typically placed between the excitation volume 108and the forward-scatter imaging system 136 to prevent the unscatteredexcitation beam from reaching the forward-scatter imaging system 136.Forward-scattered light passing around the edges of the beam block 138is collected and focused onto the active element of the forward-scatterdetector 140. A bandpass filter 142 is typically inserted between theexcitation volume 108 and the forward-scatter detector 140 to transmitlight at the excitation wavelength and to block light at otherwavelengths.

In the typical sheath-flow cytometer system 100, the excitation volume108 is defined by the intersection of a tightly focused laser-excitationsource and a sample-fluid stream with a typical diameter of a fewmicrons. Light that is scattered and emitted from the sample particlesemanates from a small excitation volume 108 that closely approximates apoint source.

Fluorescent light is typically generated by probe molecules (organic dyemolecules, for example) that are biochemically attached to certainsample particles or to specific structures within certain sampleparticles before they are introduced into the flow. Probe molecules aretypically strong absorbers of excitation light and efficiently convertabsorbed light energy to fluorescent emission. A red shift (or Stokesshift) of the fluorescent-light wavelength with respect to theexcitation-light wavelength allows the fluorescent light to be separatedfrom the excitation light with a conventional transmission filter orgrating. Fluorescent photons are typically emitted within a fewnanoseconds after the absorption of a photon from the excitation beam.This delay is short compared to the time required for a particle totravel through the excitation volume 108 in the typical sheath-flowcytometer system 100.

In certain applications, probe molecules with different emission spectraor different excitation spectra may be bonded to different types ofsample particles or to different structures within a single type ofsample particle. By measuring the amplitude of the fluorescent-lightpulses at different wavelengths, it is possible to make simultaneousmeasurements on a single particle and to differentiate signals that areproduced by different sample particles or structures.

Scattered excitation light may be used to discriminate among differentsample particle types. The amount of light that is scattered at smallangles to the propagation axis of the excitation beam varies withparticle size while large-angle scattering increases with particlegranularity and with other parameters. Certain particle species may bediscriminated by measuring the ratio of small-angle to large-anglescattering.

The shape and amplitude of the light pulses that reach the detectors126, 130, 132, 140 are determined by the optical properties of theparticles, by the particle velocities, by the dimensions of theexcitation volume 108, by properties of the light source 106, and by theoptical design of the collection optical systems 120, 136 and excitationoptical system 116. The optical properties of the particles aredependent on their size, shape, and transparency in addition to theabsorption and emission characteristics of any probes that are attachedto the particles. Strongly absorbing probes with a high quantum yieldfor fluorescent emission typically generate pulses of maximum amplitude.

In a typical application, at least one detector 126, 130, 132, 140receives a light pulse when a particle is illuminated by the excitationbeam. Each interaction between a particle and the excitation beam isknown as an “event.” In the ideal case, a particle can be identifiedfrom the characteristics of the detector pulses that are generatedduring an event. For example, it is possible to count and todiscriminate among monocytes, granulocytes, and lymphocytes in a sampleby measuring the relative magnitude of the small- and large-anglescattering signals. Errors are introduced into theparticle-identification process by deviations from smooth laminar flow,by spatial variations in particle velocity and collection efficiency,and by the simultaneous illumination of multiple particles.

In a typical capillary tube, the flow velocity has a parabolicdistribution with the greatest velocity in the tube center. Theparabolic distribution is nearly flat (radial derivative near zero) nearthe tube axis, and particles traveling in a region near the axis haveapproximately the same velocity. In capillary-flow cytometers, particlestraveling near the wall of the tube have a significantly lower velocityand produce longer pulses than those traveling near the center.Deviations from the laminar-flow condition (turbulent flow) lead tounpredictable, time-dependent pulse-shape variations.

While the vast majority of commercial and research flow cytometersutilize a sheath-flow cell as shown in FIG. 1 and described above, someflow cytometers (e.g., those manufactured by Guava Technologies) arebased on an alternative, and simpler, flow-cell design in which thesample fluid completely fills a square capillary cell. FIGS. 2A and 2Bare cross sections of representative sheath-flow and capillary-flowcells, respectively. Conventional sheath-flow cytometers are described,for example, in U.S. Pat. Nos. 4,662,742 and 4,745,285. Astate-of-the-art capillary-flow system is described in U.S. PatentPublication 2002/0028434 A1.

In the sheath-flow cell of FIG. 2A, the particle-containing sample fluid200 is confined to a region near the capillary axis by a clear sheathfluid 202. As described above, the sample 200 is introduced into thesheath fluid 202 by a specially designed core injector 102, and the twofluids 200, 202 flow through the cell under a positive pressure providedby the sheath-fluid 204 and sample-fluid 212 inlets. Between the coreinjector 102 and the excitation volume 108, the combined sheath 202 andsample 200 fluids travel through a tapered, neckdown region 110 wherethe flow cross-section is reduced. This reduction in the diameter of theflow-tube 104 increases the flow velocity and reduces the diameter ofthe sample 200 and sheath 202 fluids. Typical diameters for the samplefluid 200 in the excitation region 108 of a sheath-flow cytometer system100 are in the range of 2 μm to 25 μm, while the diameter of the sheathfluid 202 is typically greater than 100 μm.

In the capillary-flow system 206 of FIG. 2B, there is no sheath fluid,and the sample fluid 200 and the excitation volume 108 fill the entirecross-section of the capillary 208. The sample fluid 200 is drawn from asample reservoir 210 by a pump (not shown) on the downstream end of thecapillary 208 and pumped through the excitation volume 108. Sampleparticles emit and scatter light at all points throughout thecross-section of the capillary 208. The cross-sectional dimension of thesample fluid 200 in the excitation region 108 is significantly largerthan the cross-sectional dimension of the sample fluid 200 in thesheath-flow system 100. For example, a typical inside edge dimension fora square capillary 208 in a capillary-flow cytometer is 100 μm.

In general, conventional sheath-flow cytometers 100 have the followingperformance advantages when compared to capillary-flow cytometers:

-   (1) The variation in flow velocity in the excitation volume 108 is    small. The sample fluid 200 is restricted to a region of the    parabolic flow-velocity distribution where the first derivative of    the particle velocity is small. This is in contrast to a    capillary-flow system where particles flow through the entire cross    section of the capillary 208.-   (2) Variations in optical-collection efficiency are small. Because    the sample fluid 200 is confined to a small region near the    flow-tube axis, the excitation volume 108 typically acts like a    fixed point source, and wall effects have a negligible effect on    pulse amplitude. This is in contrast to conventional capillary-flow    instruments where wall effects typically cause significant,    position-dependent variations in pulse amplitude.-   (3) The smaller excitation volume 108 in sheath-flow instruments    makes it possible to use a collection lens with a higher numerical    aperture. It also reduces the background noise level and the    probability of simultaneously illuminating multiple particles.

For many measurements, however, capillary systems provide adequatemeasurement accuracy and offer the following advantages over sheath-flowsystems 100:

-   (1) Capillary systems are cheaper and less complex. Sheath-flow    cells are complex, expensive, and difficult to align properly.    Capillary-flow cells are simpler, cheaper, and less prone to    misalignment.-   (2) The sample fluid 200 is drawn through the capillary 208 by a    pump, thereby facilitating the direct measurement of particle    concentration in the sample fluid 200. In a sheath-flow cytometer    100, the sample 200 and sheath 202 fluids are injected into the flow    tube 104 under pressure, and particle concentrations are typically    measured indirectly by introducing a sample fluid 200 with a known    particle concentration into the system.-   (3) The sheath fluid 202 and associated fluidics are eliminated The    simpler fluidics of a capillary-flow instrument offer significant    cost savings for certain common measurements where reductions in    measurement accuracy are acceptable.

According to Shapiro (Practical Flow Cytometry, 4^(th) Edition, Wiley,Hoboken, 2003), “the measurement precision of a cytometer is routinelycharacterized by accumulating a distribution of measured values offluorescence or light scattering intensities from ‘nearly identicalparticles’ and computing the coefficient of variation (CV), which,expressed as a percentage, is 100 times the standard deviation for themeasurement divided by the arithmetic mean, or average.” Smaller CVs areassociated with increased accuracy.

In a typical measurement, a count is increased whenever the amplitude ofa pulse from a detector exceeds a predetermined threshold value.Variations in the pulse amplitudes produced by identical particles leadto counting errors and thus to an undesirable increase of the CV for ameasurement. CVs in conventional capillary-flow cytometers typicallyexceed those of sheath-flow instruments 100 because of thecapillary-flow cytometers' larger excitation volumes 108 and because ofthe emission of light from particles far removed from the capillaryaxis.

The CVs for measurements made with a capillary-flow cytometer may beimproved by concentrating the sample particles in a small region nearthe capillary axis. U.S. Pat. No. 6,710,871, for example, describes acapillary-flow cytometer system in which a magnetic field is used toforce magnetically-charged particles to flow within a restrictedcross-sectional area of the capillary 208.

The CVs of measurements made with a capillary-flow cytometer may also beimproved (that is, decreased) through the use of digitalsignal-processing algorithms for the determination of pulse velocity andthe real-time identification of pulses that are generated by thesimultaneous illumination of two or more particles. In comparison tosheath-flow instruments 100, the probability of simultaneouslyilluminating two particles is increased due to the larger excitationvolume 108. Improved doublet detection in capillary-flow instruments maybe accomplished by applying a combination of velocity-determiningalgorithms and conventional, sheath-flow methods as outlined in “DoubletDiscrimination in DNA Cell-Cycle Analysis,” by R. P. Wersto, et. al.,Cytometry, 46:296-306 (2001).

The optical collection system for large-angle light emission that isused in a conventional capillary-flow cytometer collects light that isemitted or scattered into a cone of angles about the collection-systemaxis. Reflection and refraction of light by the walls of the flow tubetypically lead to the collection of different amounts of light fromidentical particles that are excited at different points within the flowtube. Variations in the amount of collected light lead to variations inthe amplitude or shape of the electronic pulses produced by thedetectors and thus to an increase in the CVs for measurements made withthe instrument.

In principle, the excitation volume 108 could be reduced by using acapillary 208 with a smaller bore, but a reduction in the diameter ofthe capillary 208 leads to an increased probability that sampleparticles will clump together and clog the capillary 208. Capillaryclogging is fatal to any measurement and places a practical lower limiton the bore dimension of a square capillary 208 that is determined bythe size of the particles.

Economical and efficient methods for reducing wall effects are unknownin the prior art.

BRIEF SUMMARY OF THE INVENTION

In view of the foregoing, the present invention provides improvedcapillaries for use in capillary-flow cytometers. The cross-sectionalshape of capillaries made according to the present invention lack pointsymmetry. This significantly reduces position dependent variations inpulse shape and amplitude caused by scattering and refraction of lightby the capillary walls. The shape of the outer and inner capillary wallslowers (improves) the measured CV.

In some embodiments, capillaries have inner side walls that are tiltedat angles with respect to the collection-system optical axis so that thewidest dimension of the inner bore is closest to the collection opticalsystem and have an outer wall closest to the collection optical systemwith a dimension large enough to minimize the contribution of outer-wallrefraction to the collected light signal. The position-sensitivevariation in collected light and the associated contribution to measuredCV can be minimized by selecting the inner-wall tilt angle andouter-wall dimension according to the numerical aperture and field-stopdiameter of the collection optical system.

Exemplary capillary embodiments include tubes with a rectangular outerwall and a trapezoidal inner wall, a rectangular outer wall and atriangular inner wall, triangular outer and inner walls, and atriangular outer wall with a trapezoidal inner wall. Some capillarieshave hemispherical or rhomboid outer walls and trapezoidal or triangularinner walls.

Further embodiments of the invention minimize the amount of scatteredexcitation light entering the collection optical system by having walldimensions in the direction parallel to the collection axis that arelarge enough to minimize interactions between the excitation beam andthe outer wall.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

While the appended claims set forth the features of the presentinvention with particularity, the invention, together with its objectsand advantages, may be best understood from the following detaileddescription taken in conjunction with the accompanying drawings ofwhich:

FIG. 1 is a schematic diagram of a conventional sheath-flow cytometersystem from the prior art;

FIG. 2A is a cross-sectional view of the nozzle and excitation volume ofa typical sheath-flow cytometer;

FIG. 2B is a cross-sectional view of the flow tube and excitation volumeof a typical capillary-flow cytometer;

FIG. 3 is a cross-sectional view of a conventional square capillary;

FIG. 4 is a schematic diagram of an exemplary optical collection systemfor large-angle light emission;

FIGS. 5A through 5G are schematic diagrams of light collection fromparticles at various positions within a square capillary;

FIG. 6 is a cross-sectional view of an exemplary capillary lacking pointsymmetry according to the present invention;

FIGS. 7A through 7G are schematic diagrams of light collection fromparticles at various positions within the exemplary capillary of FIG. 6;

FIG. 8 is a table comparing the percentage of light captured in acapillary-flow cytometer when using three different capillaries; and

FIG. 9 shows cross-sectional views of three exemplary capillarieslacking point symmetry according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Turning to the drawings, wherein like reference numerals refer to likeelements, the present invention is illustrated as being implemented in asuitable environment. The following description is based on embodimentsof the invention and should not be taken as limiting the invention withregard to alternative embodiments that are not explicitly describedherein.

Position-dependent variations in pulse shape and in amplitude caused byscattering and refraction of light by the capillary walls in prior artcapillary-flow cytometers may be significantly reduced through the useof improved capillary designs. In contrast to the square and roundcapillaries of the prior art, the cross-sectional shapes of the improvedcapillaries lack point symmetry. The replacement of a conventionalsquare capillary with an asymmetric capillary embodying the presentinvention minimizes wall effects. CVs for fluorescent countingmeasurements performed by capillary-flow cytometers incorporating theimproved capillaries are expected to be principally limited by volumeeffects due to doublet and background light rather than by wall effects.The improved devices attain CVs approximately less than two times largerthan those obtained with conventional sheath-flow cytometers. Thisperformance may be contrasted to conventional capillary-flow instrumentswith CVs that are five to ten times greater than the CVs of sheath-flowinstruments.

Embodiments of the present invention may advantageously be mass-producedusing wafer-scale techniques that are known in the prior art. Forexample, capillaries with a wide range of inner-bore geometries may bemanufactured using high precision powder blasting and optical bondingtechniques, photolithography, glass molding, and stamping. They may alsobe produced using conventional glass-drawing techniques.

To illustrate the inherent limitations of the prior art symmetriccapillary designs, consider the representative square capillary 208shown in FIG. 3. This square capillary 208 is similar to those used inGuava Technologies' commercial capillary-flow cytometers. As seen in theFigure, both the outer side wall 300 and the inner side wall 302 (whichdefines the bore 304 of the capillary 208) are roughly square.

Fluorescent emission from sample particles in the capillary 208 of FIG.3 may be imaged into a single photomultiplier-tube detector 126 with therepresentative optical collection system for large-angle light emission120 of FIG. 4. In this system, light is collected by an AR-coated,molded glass aspherical lens 400 that has a numerical aperture of 0.50and an effective focal length of 8.00 mm. The aspherical lens 400 istypically positioned at a distance of 5.8 mm from the wall of thecapillary 208. The distance from the aspherical lens 400 to thephotomultiplier-tube detector 126 is approximately 150 mm. A relay lens402 is positioned approximately 90 mm from the aspherical lens 400. Thesystem performance is unaffected by the position of the longpass filters404 and by the position of the bandpass filter 134. In addition to therepresentative optical system of FIG. 4, light from capillariesaccording to the present invention may be collected using alternativeoptical systems known in the flow-cytometry art.

FIGS. 5A through 5G illustrate light collection from identicalfluorescent particles that are located at different positions within thebore 304 of the square capillary 208 of FIG. 3. The particles areidentically illuminated in each figure. In the following table, aparticle's position is specified with respect to a rectangularcoordinate system that has an origin located at the center of symmetryof the capillary 208. The x-axis is directed horizontally, parallel tothe outer side wall 300 of the capillary 208, and the y-axis is directedin the orthogonal, vertical direction. The focal line of the excitationbeam is coincident with the y-axis of the coordinate system. Individualray paths are shown as lines in FIGS. 5A through 5G, and the collectedrays are shaded gray.

Particle's Position FIG. Within the Capillary Bore 5A Center: (0, 0) 5B(0, −45 μm) 5C (0, 45 μm) 5D (−45 μm, 0) 5E (45 μm, 0) 5F (−35 μm, 35μm) 5G (35 μm, −35 μm

In FIG. 5A, the particle is located at the center of the bore 304 of thesquare capillary 208. Rays are emitted in all directions and arerefracted by the air-glass interface at the outer side wall 300 of thecapillary 208. Refraction at the interface between the sample and theinner side wall 302 of the capillary 208 is negligible because thedifference in the refractive indices is small. Rays that are shaded grayat the top of FIG. 5A are collected by the molded glass aspherical lens400 (see FIG. 4) and are directed to one or more detectors 126. Asignificant feature of light collection from a point at the center ofthe capillary 208 is the straightforward effect of refraction at theair-glass boundary: This refraction narrows the cone of collected rayswithin the capillary 208, effectively decreasing the numerical apertureof the collection optical system 120 (see FIGS. 1 and 4).

FIGS. 5B through 5G show light collection from particles that aredisplaced from the center of the bore 304 of the capillary 208. Acareful examination of FIGS. 5B through 5G shows that the amount oflight collected from these off-axis points is influenced by two effects:

-   -   (1) refraction at the air/glass boundary at the outer side wall        300 of the capillary 208 and    -   (2) small-angle reflection at the interface between the sample        and the inner side wall 302 of the capillary 208.        Compared to an on-axis emitter, outer-wall refraction leads to        an increase in the number of rays of light collected from        certain off-axis points, while reflection from the inner side        wall 302 decreases the number of collected rays. The relative        magnitude of these effects and their ultimate effect on measured        CV are functions of the optical system numerical aperture, the        field-stop diameter, and the shape of the capillary 208. The        position-dependent variation in the number of collected rays has        a deleterious effect on the precision of a capillary-flow        cytometer. Variations in collection efficiency increase the        standard deviation for measurements performed on identical        particles and deleteriously increase the CV.

According to the present invention, the measured CV may be minimized bychanging the shape of the inner and outer side walls of the capillary.The optimal capillary dimensions depend on the numerical aperture andfield-stop diameter of the collection optical system. In someembodiments of the present invention, capillaries have inner side wallsthat are tilted at angles with respect to the collection system opticalaxis so that the widest dimension of the inner bore is closest to thecollection optical system. In addition, the capillary's outer side wallclosest to the collection optical system has a dimension large enough tominimize the contribution of outer-wall refraction to the collectedlight signal. The position-sensitive variation in collected light andthe associated contribution to the measured CV may be advantageouslyminimized by selecting the inner wall tilt angle and the outer walldimension based on the numerical aperture and field-stop diameter of thecollection optical system.

FIG. 6 shows an improved capillary 600 embodying aspects of the presentinvention. The capillary 600 minimizes the positional variation in theamount of light scattered or emitted by sample particles and collectedby an optical system with a numerical aperture of 0.5. The capillary 600has a rectangular outer side wall 602 that is 680 μm wide in the x-axisdirection and 300 μm wide in the y-axis direction. The cross section ofthe inner bore 604 is trapezoidal, and the bore 604 is centered withinthe rectangular outer side wall 602. The long side of the trapezoidmeasures 150 μm, and the short side is 50 μm long. The, long and shortsides are separated by a perpendicular distance of 100 μm.

FIGS. 7A through 7G show light collection from identical particles thatare displaced from the center of the bore 604 of the capillary 600 ofFIG. 6 by distances equal to those in FIGS. 5A through 5G, respectively.(See Table below.) The effects of outer-wall refraction and ofsmall-angle reflection are significantly reduced by the trapezoidalembodiment and by the rectangular outer wall shape. In particular,compare FIGS. 5B and 7B. In FIG. 5B, light refracted from the side facesof the capillary 208 is directed toward the optical collection system(not shown but toward the top of FIG. 5B). In contrast, in FIG. 7B theextended face of the capillary 600 that is closest to the optical systemprevents this light from reaching the optical collection system. Theresult is that substantially all light reaching the optical collectionsystem in FIGS. 7A through 7G passes through this front face of thecapillary 600. Here, “substantially all” means that 75% or more of thelight that reaches the optical collection system passes through thefront face of the capillary 600. Higher percentages are preferable and,in some embodiments, the percentage exceeds 90%.

Particle's Position FIG. Within the Capillary Bore 7A Center: (0, 0) 7B(0, −45 μm) 7C (0, 45 μm) 7D (−45 μm, 0) 7E (45 μm, 0) 7F (−35 μm, 35μm) 7G (35 μm, −35 μm

FIG. 8 summarizes the results of numerical ray-tracing calculations forlight-collection efficiency in FIGS. 5A through 5G (top row) and inFIGS. 7A through 7G (bottom row) under the assumptions that theparticles are uniformly distributed inside the capillaries and that thecapillaries are identically illuminated. The center row summarizes theresults of an additional calculation for the case of a capillary with asquare inner bore and a rectangular outer bore. Columns 2 through 8 showthe percentage of light collected by an optical system from particles atdifferent positions inside the bore. Column 9 shows the average ofcolumns 2 through 8, and column 10 shows the value of the CV calculatedfrom data in the other columns. Comparing the bottom row with the othertwo rows, the variation in collected light and the corresponding CVs aresignificantly reduced by the capillary 600 made according to aspects ofthe present invention.

Conventionally, the measured CVs of a prior art capillary cytometer witha square capillary may be reduced by placing apertures in the collectionoptical system to block a portion of the side-wall refracted light.Unfortunately, the insertion of corrective apertures has the undesirableeffect of reducing the amount of light collected, thereby reducing thesensitivity. Aperture-corrected systems impose tighter dimensionaltolerances on the capillary and are sensitive to the relative positionof the aperture and capillary, often requiring active alignment foroptimal results. In the best case, the CVs of the measurements made withan aperture-corrected capillary-flow cytometer are two to three timesgreater than the theoretical CVs of measurements made with acapillary-flow instrument incorporating the trapezoidal embodiment ofFIG. 6.

While offering significant improvements in the performance of theoptical collection system for large-angle light emission, the embodimentof FIG. 6 has little effect on sample excitation and on the measurementof small-angle forward scatter. There is negligible refraction at thesmall index step at the inner sample/glass interface. The contributionsof reflection at the inner wall and refraction at the outer wall on theamount of light reaching the forward scatter detector are comparable inmagnitude to those observed in a conventional square capillary.

Some alternative embodiments of the invention share the followingproperties with the FIG. 6 embodiment.

-   -   (1) The dimension of the outer wall in the direction        perpendicular to the axis of the optical collection system for        large-angle light emission is chosen in such a way that signal        contributions from outer-side-wall refraction and corner        refraction are minimized.    -   (2) The inner side walls are angled with respect to the        collection system axis to minimize the contribution of        small-angle reflection and the corresponding position-sensitive        variations in the collected light signal. The magnitude of the        inner-side-wall angle is dependent on the numerical aperture of        the collection optical system, being greater than 20 degrees for        a system with a numerical aperture of 0.5 and greater than 5        degrees for a system with a numerical aperture of 0.1.

A few alternative embodiments incorporating these design principles areshown in FIG. 9. They include designs with trapezoidal outer and innerwalls (900), a rectangular outer wall and a triangular inner wall (902),and triangular outer and inner walls (904). These alternatives are notexclusive but should be considered representative of capillary designsembodying the present invention.

Further embodiments of the invention minimize the amount of refractedexcitation light entering the collection optical system by having walldimensions in the direction parallel to the collection axis that arelarge enough to minimize interactions between the excitation beam andthe outer wall. For example, with an illuminating laser beam diameter of0.7 mm at the 1/e power points, background light levels may be minimizedby capillary designs in which the dimension of the outer capillary wallin the direction parallel to the large-angle collection axis is greaterthan 0.7 mm. Advantageously, the amount of background light reaching thedetector may be further reduced by decreasing the area of the inner boreto the limit imposed by clogging.

Capillaries embodying the features of the present invention may bemass-produced using wafer-scale techniques. Such techniques are, forexample, practiced commercially by Anteryon B V, P.O. Box 33, 5600 AAEindhoven, The Netherlands. To fabricate the capillary 600 of FIG. 6,for example, a large number of trapezoidal bores are fabricated in afirst glass plate using Anteryon's high precision powder blastingtechniques. After the trapezoidal shapes are formed in the first plate,hollow, trapezoidal cores are formed by bonding the first plate to asecond glass plate. The thickness of the bonded plates is chosen in sucha way that the core position and thickness in the y-direction matchthose in FIG. 6. Appropriate dimensions in the x-direction are obtainedby sawing the bonded plates. Using these techniques, a large number ofcapillaries are fabricated in a single process, thereby minimizing thecost of an individual capillary. This method of capillary manufactureallows the fabrication of a wide range of different core geometries andcould be modified to form structures that require the blasting of bothplates.

In view of the many possible embodiments to which the principles of thepresent invention may be applied, it should be recognized that theembodiments described herein with respect to the drawing figures aremeant to be illustrative only and should not be taken as limiting thescope of the invention. Those skilled in the flow-cytometry oroptical-fabrication arts will realize that the invention may bepracticed using capillary designs other than those shown in FIGS. 6 and9. Such capillary designs would simultaneously reduce the contributionsof outer-wall refraction and inner-wall reflection to the large-anglecollected light signal but may have alternative outer- and inner-wallgeometries. For example, capillaries with hemispherical or rhomboidouter walls and trapezoidal or triangular inner walls clearly embody thefeatures of the invention. Therefore, the invention as described hereincontemplates all such embodiments as may come within the scope of thefollowing claims and equivalents thereof.

1. A capillary for use in a capillary-flow cytometer, the capillarycomprising: a wall defining a bore extending in a longitudinaldirection, the wall further comprising: a substantially non-reflectiveinner wall surface defining a cross-sectional shape of the bore, thecross-sectional shape of the bore being traverse to the longitudinaldirection of the bore and lacking point symmetry; and an outer wallsurface defining a cross-sectional shape of the wall that is traverse tothe longitudinal direction; wherein the cross-sectional shape of thewall is the same as the cross-sectional shape of the bore; and whereinthe capillary is configured so that upon installation in acapillary-flow cytometer, substantially all light collected by anoptical collection system of the capillary-flow cytometer passes througha side of the outer wall surface that is closest to the opticalcollection system.
 2. The capillary of claim 1 wherein thecross-sectional shapes defined by the outer and inner wall surfaces aretriangles.
 3. A capillary for use in a capillary-flow cytometer, thecapillary comprising: a wall defining a bore extending in a longitudinaldirection, the wall further comprising: an outer wall surface defining across-sectional shape of the wall, the cross-sectional shape of the wallbeing traverse to the longitudinal direction and selected from the groupconsisting of a hemisphere and a rhomboid; and a substantiallynon-reflective inner wall surface defining a cross-sectional shape ofthe bore, the shape of the bore being traverse to the longitudinaldirection of the bore and selected from the group consisting of atrapezoid and a triangle; wherein the capillary is configured so thatupon installation in a capillary-flow cytometer, substantially all lightcollected by an optical collection system of the capillary-flowcytometer passes through a side of the outer wall surface that isclosest to the optical collection system.
 4. A capillary-flow cytometercomprising: a capillary comprising a wall having inner and outer wallsurfaces and extending in a longitudinal direction, the inner wallsurface defining a bore also extending in the longitudinal direction andhaving a cross-sectional shape lacking point symmetry; a fluidics systemfor creating a pressure differential in the bore in the longitudinaldirection and transporting a particle-containing sample fluid throughthe capillary; an excitation optical system configured for illuminatinga portion of the capillary; an optical collection system configured forcollecting light from the sample fluid as it travels through theilluminated portion capillary; an optical detector configured fordetecting light collected by the optical collection system; and a dataacquisition and analyzer system for analyzing signals from the opticaldetector; wherein the capillary is configured so that substantially alllight collected by the optical collection system passes through an areaof the outer wall surface of the capillary that is closest to theoptical collection system and the inner wall surface is substantiallynon-reflective.
 5. The capillary-flow cytometer of claim 4 wherein across-sectional shape defined by the outer wall surface of the capillaryand traverse to the longitudinal direction of the wall is of the sameshape as the cross-sectional shape defined by the inner wall surface ofthe capillary.
 6. The capillary-flow cytometer of claim 5 wherein thecross-sectional shapes defined by the outer and inner wall surfaces ofthe capillary are triangles.
 7. The capillary-flow cytometer of claim 4wherein a cross-sectional shape defined by the outer wall surface of thecapillary and traverse to the longitudinal direction of the wall differsin shape from the cross-sectional shape defined by the inner wallsurface of the capillary.
 8. The capillary-flow cytometer of claim 7wherein the cross-sectional shape defined by the outer wall surface ofthe capillary is a rectangle; and wherein the cross-sectional shapedefined by the inner wall surface of the capillary is a trapezoid. 9.The capillary-flow cyrometer of claim 7 wherein the cross-sectionalshape defined by the outer wall surface of the capillary is a rectangle;and wherein the cross-sectional shape defined by the inner wall surfaceof the capillary is a triangle.
 10. The capillary-flow cytometer ofclaim 7 wherein the cross-sectional shape defined by the outer wallsurface of the capillary is a triangle; and wherein the cross-sectionalshape defined by the inner wall surface of the capillary is a trapezoid.11. The capillary-flow cytometer of claim 7 wherein the cross-sectionalshape defined by the outer wall surface of the capillary is selectedfrom the group consisting of a hemisphere and a rhomboid; and whereinthe cross-sectional shape defined by the inner wall surface of thecapillary is selected from the group consisting of a trapezoid and atriangle.
 12. The capillary-flow cytometer of claim 4 wherein the innerwall surface of the capillary is tilted with respect to an axis of theoptical collection system so that a widest dimension of the bore of thecapillary is closest to the optical collection system.
 13. A capillaryflow cytometer comprising: a capillary having a wall extending in alongitudinal direction to define a bore for supporting flow of aparticle-containing sample fluid, the wall having inner and outer wallsurfaces, the inner wall surface defining a cross-sectional shape of thebore such that the cross-sectional shape lacks point symmetry; afluidies system that transports the particle-containing sample fluidthrough the bore through the creation of a fluid pressure differential;an excitation optical system for passing light through a first side ofthe capillary wall and illuminating the sample fluid as it passesthrough a portion of the capillary; an optical collection system forcollecting light from the illuminated sample fluid, whereinsubstantially all of the collected light passes through a second side ofthe wall; an optical detector for detecting light collected by theoptical collection system; and a data acquisition and analyzer systemfor analyzing signals from the optical detector.
 14. The flow cytometerof claim 13 wherein the excitation light is substantially transmitted bythe first side of the wall.
 15. The capillary of claim 13 in which thewall is formed from a material that is selected from the groupconsisting of glass, plastic and fused silica.
 16. The capillary ofclaim 13 wherein the second side has a shape that is a section of ageometric figure selected from the group consisting of a circle, aparabola, and an ellipse.
 17. A capillary for use in a capillary-flowcytometer, the capillary comprising: a wall defining a bore extending ina longitudinal direction, the wall further comprising: an outer wallsurface having a side, the side having a shape that is a section of ageometric figure selected from the group consisting of a circle, aparabola, and an ellipse; and a substantially non-reflective inner wallsurface defining a cross-sectional shape of the bore, thecross-sectional shape of the bore being traverse to the longitudinaldirection and lacking point symmetry; wherein the capillary isconfigured so that upon installation in a capillary-flow cytometer withthe side of the outer wall surface closest to an optical collectionsystem of the capillary-flow cytometer, substantially all lightcollected by the optical collection system passes through the side. 18.A capillary for use in a capillary-flow cytometer, the capillarycomprising: a wait defining a bore extending in a longitudinaldirection, the wall further comprising: a substantially non-reflectiveinner wall surface defining a trapezoidal cross-sectional shape of thebore traverse to the longitudinal direction; and an outer wall surfacedefining a triangular cross-sectional shape of the wall traverse to thelongitudinal direction; wherein the capillary is configured so that uponinstallation in a capillary-flow cytometer, substantially all lightcollected by an optical collection system of the capillary-flowcytometer passes through a side of the outer wall surface that isclosest to the optical collection system.